Inhibition of spleen tyrosine kinase decreases donor specific antibody levels in a rat model of sensitization

Antibody mediated rejection is a major cause of renal allograft loss. Circulating preformed donor specific antibodies (DSA) can result as a consequence of blood transfusion, pregnancy or prior transplantation. Current treatment strategies are limited due to partial or transient efficacy, adverse side-effects or patient unsuitability. Previous in vivo studies exploring autoimmune diseases have shown that spleen tyrosine kinase (SYK) signalling is involved in the development of pathogenic autoantibody. The role of SYK in allogenic antibody production is unknown, and we investigated this in a rodent model of sensitization, established by the transfusion of F344 whole blood into LEW rats. Two-week treatment of sensitized rats with selective SYK inhibitor fostamatinib strongly blocked circulating DSA production without affecting overall total immunoglobulin levels, and inhibition was sustained up to 5 weeks post-completion of the treatment regimen. Fostamatinib treatment did not affect mature B cell subset or plasma cell levels, which remained similar between non-treated controls, vehicle treated and fostamatinib treated animals. Our data indicate fostamatinib may provide an alternative therapeutic option for patients who are at risk of sensitization following blood transfusion while awaiting renal transplant.

Antibody mediated rejection is a major cause of renal allograft loss. Circulating preformed donor specific antibodies (DSA) can result as a consequence of blood transfusion, pregnancy or prior transplantation. Current treatment strategies are limited due to partial or transient efficacy, adverse side-effects or patient unsuitability. Previous in vivo studies exploring autoimmune diseases have shown that spleen tyrosine kinase (SYK) signalling is involved in the development of pathogenic autoantibody. The role of SYK in allogenic antibody production is unknown, and we investigated this in a rodent model of sensitization, established by the transfusion of F344 whole blood into LEW rats. Two-week treatment of sensitized rats with selective SYK inhibitor fostamatinib strongly blocked circulating DSA production without affecting overall total immunoglobulin levels, and inhibition was sustained up to 5 weeks post-completion of the treatment regimen. Fostamatinib treatment did not affect mature B cell subset or plasma cell levels, which remained similar between non-treated controls, vehicle treated and fostamatinib treated animals. Our data indicate fostamatinib may provide an alternative therapeutic option for patients who are at risk of sensitization following blood transfusion while awaiting renal transplant.
Antibody mediated rejection (AMR) represents a significant barrier to allograft survival. The presence of preformed alloreactive donor specific antibodies (DSA) can arise from exposure to cells originating from other individuals via pregnancy, prior transplantation and blood transfusion 1 .
There is abundant research implicating the pathogenic role of DSA directed against polymorphic human leukocyte antigen class I (HLA I) and HLA class II (HLA II) in multiple organs. Non-HLA targets also play a crucial role in allograft rejection and DSA targets include minor histocompatibility molecules such as major histocompatibility complex (MHC) class I chain-related protein A (MICA) and major histocompatibility complex (MHC) class I chain-related protein B (MICB) 2 . Other commonly reported non-HLA targets are expressed on epithelial cells and endothelial cells 3,4 .
There are two main mechanisms of allograft damage by DSA. The classical complement pathway can be activated by DSA binding to target antigen on endothelium, with generation of complement components which recruit inflammatory cells to the graft or directly damage the graft through the formation of the membrane attack complex 5 . In the process of antibody-dependent cell mediated cytotoxicity (ADCC), effector cells bearing Fcγ receptors (FcγR) can interact with the crystalline fragment (Fc) of bound DSA on endothelium and trigger lysis of target cells 6 .
Over 40% of patients awaiting kidney transplant in the United Kingdom are presensitized, with a reported median wait time of 6.1 years, which is double that of non-sensitized patients 7 . Current desensitization strategies include plasmapheresis and intravenous immunoglobulin, with or without immunosuppression using the B cell depletion-agent rituximab 8,9 . However, these strategies are limited and have partial or transient efficacy, in addition to their adverse effects and unsuitability for patients with certain comorbidities 10 . It is crucial therefore to identify new, safer and more effective therapeutic targets for desensitization of patients.
Spleen tyrosine kinase (SYK) is a cytosolic non-receptor tyrosine kinase mainly expressed in haemopoietic cells. Activation of SYK and subsequent signal transduction is initiated downstream of classical immunoreceptors www.nature.com/scientificreports/ including FcγR and the B cell receptor (BCR). In the B cell, SYK signalling plays a critical role in B cell maturation and effector functions [11][12][13][14] . Increasing numbers of studies have targeted SYK for the treatment of immune and inflammatory diseases, and it has potential efficacy in the treatment of presensitized patients [15][16][17][18] . Fostamatinib is a small molecule SYK inhibitor, and through its active metabolite R406 has previously shown efficacy in the treatment of experimental autoimmune glomerulonephritis in rats, where treatment resulted in attenuation of autoantibody production 19 .
Here, in a rat model of sensitization, we demonstrate that fostamatinib treatment is able to prevent the production of allogenic antibody, even after cessation of treatment, maintaining overall non-allogenic immunoglobulin levels, whilst having no depletory effect on the levels of plasma cell or B cell populations.

Materials and methods
Animals and transfusions. Eight-week old LEW.Crl RT1 I (LEW) and F344/DUCrl RT1 lv (F344) male rats were purchased from Charles River UK Ltd (Margate, UK) and maintained in a pathogen-free animal facility at the Central Biomedical Services unit, Hammersmith Hospital Campus, Imperial College London. LEW received 800 µL heparinised whole blood from F344 via an intravenous route. All animal studies were licensed by the Home Office Science Unit. Studies and procedures were approved by Imperial College London Research Ethics committee and carried out in accordance with the regulations of the UK Animals (Scientific Procedures) Act (1986) and ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.
Treatments. SYK specific inhibitor fostamatinib was provided by Rigel Pharmaceuticals (South San Francisco, CA, USA). Fostamatinib or vehicle (0.1% carboxymethylcellulose) was administered in sensitized animals by oral gavage twice daily for a period of 14 days at 40 mg/kg from either 24 h or 7 days post-transfusion.
Detection of total serum immunoglobulin. Serum levels of total IgG and IgM were detected with Enzyme Linked Immunosorbent Assay (ELISA) from ThermoFischerScientific. Serum was diluted by 1:10,000 (IgM) or 1:50,000 (IgG).
Statistical analyses. Data were analysed with GraphPad Prism 8.0 (GraphPad Software, Inc, La Jolla, CA) and are displayed as the mean ± SEM using a Mann-Whitney U test as appropriate. A P < 0.05 was defined as significant.

Results
Transfusion results in the production of donor specific antibody. A rat model of sensitization was established by whole blood transfusion between F344 and LEW rats. T lymphocyte (CD3 + ) flow crossmatch analysis showed that transfusion between these strains elicited an alloantibody response. An initial peak in allogenic IgM levels 7 days post-transfusion (Fig. 1A), was followed by isotype switching to IgG (Fig. 1B) where levels peaked at day 17. Additionally, all DSA IgG isotypes IgG1 ( Treatment with fostamatinib inhibits allogenic antibody production. Following establishment of a sensitized model, the role of fostamatinib in DSA production was investigated. Sensitized rats were treated with fostamatinib 24 h after blood transfusion. Flow crossmatch analysis demonstrated that SYK inhibitor treatment was able to block production of significant levels of IgM ( Fig Total circulating IgG and IgM levels are not affected by fostamatinib. As fostamatinib prevented the formation of an alloantibody response, serum was analysed for total circulating IgM (Fig. 3A) and IgG levels (Fig. 3B). These levels remained comparable between treatment groups.  . 4A,B), switched cells (CD45R + CD27 + IgD − ) (Fig. 4C,E) and non-switched cells (CD45R + CD27 + IgD + ) (Fig. 4D,E), were detected at similar levels in both treatment groups compared to control rats. Given that DSA production was profoundly inhibited by fostamatinib, effects on numbers of splenic plasma cells were evaluated between treatment groups and compared to control non-sensitized LEW rats (Fig. 4F,G). Unlike DSA production, plasma cell numbers (CD138 + CD45R − ) were not affected by fostamatinib therapy and both treatment groups remained at similar levels compared to non-treated non-sensitized animals.
Fostamatinib prevented DSA levels rising 5 weeks after termination of treatment. Following potent inhibition of DSA levels with fostamatinib treatment, we wanted to evaluate if this suppression was transient or maintained. Sensitized rats were treated with fostamatinib for the 2-week regimen as described previously and, upon completion of the treatment course, rats were left untreated for 5 weeks with weekly venesection for measurement of DSA levels. T lymphocyte crossmatch analysis of serum showed that 2-week fostamatinib treatment was able to maintain suppression of formation of DSA IgM (Fig. 5A) and IgG (Fig. 5B) up to 5 weeks after completion of the treatment regimen. www.nature.com/scientificreports/ Delayed fostamatinib treatment prevented IgG DSA reaching levels found in vehicle treated rats. To assess the efficacy of fostamatinib treatment initiated at a later timepoint in sensitized rats, the fostamatinib treatment regimen was delayed until 7 days post-transfusion. Results showed that fostamatinib did not block allogenic IgM production and circulating levels were comparable between treatment groups (Fig. 6A). However allogenic IgG levels were significantly reduced from day 14 in fostamatinib treated rats (Fig. 6B). Investigating this further, IgG subsets were measured, demonstrating variable results (Fig. 6C-F). Levels of IgG2b, IgG1 and IgG2a showed significant blocking of alloantibody production from days 14, 17 and 21 respectively. IgG2c showed no difference between treatment groups at any time point.

Discussion
In this study, we demonstrate for the first time that SYK inhibition with fostamatinib significantly prevents allogenic DSA production in a rat model of sensitization. Fostamatinib treatment initiated 24 h post-transfusion for the treatment regimen of 2 weeks was effective at preventing production of both allogenic IgG and IgM, and had www.nature.com/scientificreports/ no depletory effects on total IgG and IgM levels. Fostamatinib treatment remained effective even 5 weeks posttermination of treatment, with significantly lower levels of IgG and IgM detected by the T cell crossmatch test at this time point. Fostamatinib was also able to partially block DSA production when treatment was implemented 7 days post-sensitisation, after the initiation of an allogenic antibody response. Our study shows that fostamatinib had a direct effect on inhibition of alloantibody without adversely affecting B cell survival. Splenic plasma cell numbers were comparable between both treatment groups. In addition to plasma cells, we examined the effect of SYK blockade on other mature B lymphocyte populations. Memory, switched and non-switched populations of B cells in the spleen remained similar between both treatment groups. Normal B cell activation and development is therefore not being affected by fostamatinib treatment, rather the production of allogenic antibody is. SYK has a well-defined role in signal transduction downstream of immunoreceptors and is critical in mediating BCR responses and FCR responses in mast cells 20 , dendritic cells 20 , macrophages 21 and neutrophils 22 . The SYK homologue zeta chain-associated protein kinase 70 (ZAP-70) 23 is the predominant signalling molecule downstream of the TCR in T cells and natural killer cells. Studies involving SYK knockout murine models have shown the critical role of SYK signalling for B cell development and maturation, where B cell maturation is arrested from progressing at the early pro-B-cell state in murine SYK knockout models 11,24,25 . Early mechanistic data has demonstrated that, in vitro fostamatinib was able to inhibit BCR responses in primary human B cells, where CD69 cell surface upregulation induced by BCR crosslinking with IgM was inhibited with fostamatinib treatment 26 .
The role of SYK in antibody production in plasma cells is unclear, as B cells from SYK knockout mice are unable to mature beyond the pro-B-cell stage 25 . In vivo models have provided some insight and indications that SYK inhibition may be an effective target for the treatment of antibody-mediated diseases. In a rodent model of anti-glomerular basement membrane disease, R406 treatment prevented the induction of disease, and inhibited circulating and deposited autoantibody production in established disease 19 . Interestingly in this model, CD45RA + cell numbers remained the same despite decrease of autoantibody. Non-obese diabetic mice were protected against developing diabetes with fostamatinib treatment in a prevention setting. Treatment was also able to delay disease progression in glucose intolerant mice, with a measured reduction in anti-glutamic-aciddecarboxylase anti-islet antibodies 27 .
Currently there are no reported data on the efficacy of fostamatinib in reduction of alloantibody production, and to our knowledge this is the first report of its kind. In contrast to our experiments, early immunotoxicology studies where rats were immunised with KLH-Ribi antigen and treated with fostamatinib, did not show reduced KLH specific IgM and IgG 28 . Differences between the effects of fostamatinib on production of alloantibody compared to autoantibody are intriguing and require further investigation. The mechanism of inhibition of DSA production by fostamatinib in our experiments is unclear. Due to the functional action of fostamatinib, it is likely that downstream signalling molecules have been blocked from phosphorylation, which in-turn has impacted DSA production. Fostamatinib inhibition of SYK prevents phosphorylation of downstream molecules, which include phospholipase Cγ1, Akt/protein kinase B, c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase 26 . Exactly which signals contribute to immunoglobulin production requires further investigation. It is possible in our experiments that fostamatinib treatment is blocking antigen presentation to B cells via follicular dendritic cells, a process required for the progression of the B cell response. Studies have also established a crucial role for SYK signalling in the induction of the antigen presentation machinery in both B cells 27 and dendritic cells 29 . In dendritic cells, SYK signalling has shown to be crucial for immunocomplex uptake and antigen presentation 20 .
In our model, blood transfusion initially induced IgM production, with peak titres approximately 7 days post sensitization. Traditionally in the context of AMR, preformed IgM antibodies have been perceived to be nonpathogenic. However emerging studies suggest that anti-HLA IgM may make important contributions to the pathogenesis of allograft loss [30][31][32][33] . In our experiments where treatment was implemented 24 h post sensitization, DSA IgM production was blocked, indicating this might be beneficial for human presensitization. Fostamatinib   35 . In humans four IgG subclasses exist, IgG1, IgG2, IgG3 and IgG4. It is widely accepted that IgG1 and IgG3 are the most pathogenic subclasses in AMR due to their ability to activate the complement cascade 36 . IgG 3 has the highest binding efficiency to complement component C1q, and IgG1 is highly effective at complement dependent cell lysis 37 . The presence of these subclasses as a result of sensitization is associated with poor graft outcome 33 . In early treatment experiments we have shown the efficacy of fostamatinib in blocking production of all IgG subtypes, thereby resulting in prevention of downstream effects of humoral immunity, suggesting this approach could be suitable in prevention of presensitization when given concomitantly to blood transfusions or possibly de novo DSA during transplantation 38 . In addition to DSA level reduction, utilizing fostamatinib to inhibit the effector phase and ultimately influencing the pathogenicity of the DSA underpins the rationale for using fostamatinib in AMR.
Fostamatinib treatment initiated 7 days post transfusion was able to significantly reduce the levels of DSA IgG detected in the CD3 + crossmatch assay at days 14, 17 and 21. Study of IgG subsets provided variable results. IgG2c levels were similar between groups, but IgG1 and the most pathogenic subsets IgG2a and IgG2b demonstrated significantly lower levels in treated rats from at day 14. It is probable that fostamatinib treatment was inhibitory soon after administration, as demonstrated by early time point experiments, however fostamatinib is unlikely to inhibit DSA IgG that has already been made. The circulating half-life of IgG is 7-25 days and it is likely that an extended follow-up time would continue to show more pronounced repression of DSA IgG levels. The ability of fostamatinib to block de novo DSA IgG production but not existing DSA IgG could explain the substantially higher DSA IgG levels measured in late treatment experiments compared to earlier treatment experiments.
In the context of blood transfusion as a sensitizing event, there is an abundance of literature associating allosensitization with higher rates of graft rejection and lower rates of graft survival 39 . Chronic anaemia is prevalent in patients with chronic kidney disease or end-stage kidney disease and avoiding transfusion is not always feasible 40 . A causal link between blood transfusions and DSA production has been identified by Hassan et al. 41 . In patients receiving blood transfusion after renal allograft transplantation, this study found direct evidence of a de novo HLA alloimmune response elicited against the blood donor, and enhanced risk of transplant specific antibody development within these patients. These factors were significantly associated with the increased risk of AMR and allograft failure. Current treatment options including early post-transplant erythropoietin therapy and cell salvage have limited efficacy 42 .
In a pre-transplant setting, current treatment strategies for sensitized patients have partial or transient efficacy and include the use of plasma exchange, anti-CD20 monoclonal antibody therapy with rituximab, or blocking macrophage FcγR with high-dose intravenous immunoglobulin 43 . Fostamatinib treatment presents a potential alternative strategy in preventing allosensitization in both pre-transplant and post-transplant blood transfusions. In our experiments, a relatively transient period of orally given treatment was able to inhibit DSA levels even up to 5 weeks post sensitization, which is significant in a clinical context as it is likely to improve patient compliance compared to more regular or invasive treatment. Our data are limited by the moderately short follow up time, and the long-term impact of SYK inhibition on plasma cell and B cell function is unknown. Further work will be needed to investigate whether treatment with fostamatinib may have a role in treatment of sensitized rats in models of experimental transplant rejection.
LEW and F344 rats are weakly histocompatible due to differing partially at both MHC I & II and non-MHC loci 44 . Transplantation of an F344 kidney into a LEW rat is a well-characterized model of chronic antibody mediated rejection, where rejection develops over the course of a few months 45 . It will be crucial to investigate the protective potential of fostamatinib in sensitized transplanted rats from developing chronic AMR. In a rat model   46 . This study explored the efficacy of SYK inhibitor GS-492429 in acute rejection, with a splenocyte transfusion for the sensitization method. The protective results are promising for our future studies in a chronic AMR rat model. www.nature.com/scientificreports/ Fostamatinib is currently FDA approved for the treatment of thrombocytopenia in adults with chronic ITP with insufficient response to other treatment. Appropriate dosing, PKA data and a good safety profile for fostamatinib are available 47 . The data presented in this study suggest that fostamatinib should be investigated for reduction of circulating allogenic antibodies in patients with chronic kidney disease needing blood transfusion and highly sensitized patients with end-stage renal disease awaiting transplant.

Data availability
The data supporting the findings of this study is available from the corresponding author upon reasonable request.